WO2010051882A1 - Utilisation d’eef1a comme biomarqueur et procédé de criblage d’inhibiteurs de metap2 - Google Patents

Utilisation d’eef1a comme biomarqueur et procédé de criblage d’inhibiteurs de metap2 Download PDF

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Publication number
WO2010051882A1
WO2010051882A1 PCT/EP2009/007102 EP2009007102W WO2010051882A1 WO 2010051882 A1 WO2010051882 A1 WO 2010051882A1 EP 2009007102 W EP2009007102 W EP 2009007102W WO 2010051882 A1 WO2010051882 A1 WO 2010051882A1
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WIPO (PCT)
Prior art keywords
metap2
meteefia
compounds
activity
concentration
Prior art date
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PCT/EP2009/007102
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English (en)
Inventor
Roland Kellner
Frank Zenke
Joerg Bomke
Original Assignee
Merck Patent Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merck Patent Gmbh filed Critical Merck Patent Gmbh
Priority to JP2011533565A priority Critical patent/JP5347028B2/ja
Priority to ES09778819T priority patent/ES2402564T3/es
Priority to CA2742737A priority patent/CA2742737C/fr
Priority to AU2009313167A priority patent/AU2009313167C1/en
Priority to US13/127,834 priority patent/US9151743B2/en
Priority to EP09778819A priority patent/EP2344665B1/fr
Publication of WO2010051882A1 publication Critical patent/WO2010051882A1/fr
Priority to IL212664A priority patent/IL212664A0/en

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the cellular sample is taken in-vivo or in-situ from a mammal to be tested.
  • the withdrawal of the cellular sample follows good medical practice.
  • Biological samples may be taken from any kind of biological species, but the sample is especially taken from a laboratory animal or a human, more preferably a rat, mouse or human, most preferably a human.
  • “allelic variant” is understood to represent the gene product of one of two or more different forms of a gene or DNA sequence that can exist at a genetic single locus.
  • Artificial fragments preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises the N-terminus of diagnostic interest consisting of at least the N-terminal 20 contiguous amino acids as derived from the sequence disclosed in SEQ ID NOs: 1 or 2, preferably at least the N-terminal 40 contiguous amino acids, more preferably at least the N-terminal 60 contiguous amino acids, highly preferably at least the N-terminal 82 contiguous amino acids.
  • the term "specific substances" as used herein comprises molecules with high affinity to at MetEEFIA in order to ensure a reliable binding.
  • the substances are preferably specific to parts of the protein. Such parts represent a restriction to those regions which are sufficient for the expression of a specific function, i.e. the provision of a structural determinant for recognition. All truncations are inevitably limited by the requirement of preserving the unique recognition. However, the parts of the gene products can be very small.
  • the substances are mono-specific in order to guarantee an exclusive and directed interaction with the chosen single target. It is particularly required that the specific substances are capable of discriminating between EEF1 A (e.g. a protein with an acetylated glycine residue at the N-terminus) and MetEEFIA (i.e. protein with a methionine residue at the N-terminus).
  • the nucleic acids can be optionally modified as phosphorothioate DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA) or aptamers.
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • spiegelmer particularly preferred nucleic acid probes to be used as MetAP2-specific substances are aptamers.
  • the specific substances can be labeled, in doing so the labeling depends on their inherent features and the detection method to be applied, i.e. the required sensitivity, ease of conjugation, stability requirements, and available instrumentation and disposal provisions.
  • the applied methods depend on the specific incubation products to be monitored and are well-known to the skilled artisan.
  • Radioactive radiation of isotopes is measured by scintillation.
  • the process of liquid scintillation involves the detection of beta decay within a sample via capture of beta emissions in a system of organic solvents and solutes referred to as the scintillation cocktail.
  • the beta decay electron emitted by radioactive isotopes such as 3 H, 14 C, 32 P, 33 P and 35 S in the sample excites the solvent molecule, which in turn transfers the energy to the solute.
  • the energy emission of the solute (the light photon) is converted into an electrical signal by a photo-multiplier tube within a scintillation counter.
  • the cocktail must also act as a solubilizing agent keeping a uniform suspension of the sample.
  • a labeling method is not particularly limited as long as a label is easily detected.
  • a "labeled specific substance” is one that is bound, either covalently through a linker or a chemical bond, or non-covalently through ionic, van der Waals, electrostatic, hydrophobic interactions or hydrogen bonds, to a label such that the presence of the MetEEFIA protein may be detected by detecting the presence of the label bound to the biomarker.
  • the antibodies are labeled with detectable moieties, which include, but are not limited to, radionuclides, fluorescent dyes, e.g. fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc., fluorescent markers, e.g. green fluorescent protein (GFP), phycoerythrin, etc., auto-quenched fluorescent compounds that are activated by tumor-associated proteases, enzymes, e.g.
  • detectable moieties include, but are not limited to, radionuclides, fluorescent dyes, e.g. fluorescein, fluorescein isothiocyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc., fluorescent markers, e.g. green fluorescent
  • Immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence. Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention. In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention.
  • antibodies are used as specific substances to MetEEFIA and the incubation products are detected by the labeling of the antibodies, preferably by ELISA, RIA, fluoro immunoassay (FIA), soluble particle immune assay (SPIA) or western blotting.
  • ELISA ELISA
  • RIA fluoro immunoassay
  • SPIA soluble particle immune assay
  • Antigens or antibodies, respectively, which are favorably labeled with fluorophores, are used in FIAs.
  • all methods for detection include intensive washing steps to separate unbound and/or non-specifically bound antigens from the MetEEFIA/antibody complex.
  • the experimental procedure of any detection method is well-known to those skilled in the art.
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate, using a radiation counter to detect radiation, such as a gamma counter for detection of 125 I, or using a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer, such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.
  • the antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate ⁇ e.g. microtiter wells), pieces of a solid substrate material or membrane ⁇ e.g. plastic, nylon, paper) and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers.
  • Such formats include protein microarrays or protein chips.
  • each discrete surface location may comprise antibodies to immobilize one or more protein markers for detection at each location.
  • Surfaces may alternatively comprise one or more discrete particles (e.g. microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more protein markers for detection.
  • Optical images viewed and optionally recorded by a camera or other recording device are optionally further processed in any of the embodiments herein, e.g. by digitizing the image and storing and analyzing the image on a computer.
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
  • One conventional system carries light from the specimen field to a cooled charge-coupled device (CCD) camera, in common use in the art.
  • a CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed.
  • the apparatus and methods of the invention are easily used for viewing any sample, e.g. by fluorescent or dark field microscopic techniques.
  • Non-limiting examples of sequence analysis include Edman sequencing, capillary array sequencing, thermal cycle sequencing, solid-phase sequencing, sequencing with mass spectrometry, such as MALDI-TOF/MS and sequencing by hybridization, preferably Edman sequencing.
  • Non-limiting examples of electrophoretic analysis include slab gel electrophoresis, such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, denaturing gradient gel electrophoresis and isoelectric focusing electrophoresis (IEF), preferably 1 D IEF. IEF analysis is especially stained by Coomassie Blue or silver.
  • Electrochemical processes and probes are also well-established and described in WO 2003/060464 A2, for example, which is incorporated by reference herein.
  • the induction and accumulation of MetEEFIA is detected by IEF along a first dimension followed by at least one method that is selected from the group of western blotting, mass spectrometry and N-terminal sequencing.
  • MetEEFIA bearing the methionine terminal residue is more positively charged at neutral pH in comparison with the constitutive acetylated isoform EEF1A, which allows the two isoforms to be separated by pi. Consequently, the antibody for western blotting does not have to be mono-specific to MetEEFIA, but any specific antibody to EEF1A in general can be applied, i.e.
  • step (c) comprises the further sub- steps of: (c 1 ) correlating an amount of signal or change in signal with a MetEEFIA concentration in the system, and (c") detecting a level of MetAP2 inhibition by comparing the MetEEFIA concentration with another MetEEFIA concentration in a cellular system being not incubated with the compounds and/or in a cellular system being incubated with a standard compound having MetAP2 inhibiting activity.
  • the cellular system of the invention is incubated with various concentrations of an identified MetAP2 inhibitor.
  • the amount of emitted signal or change in signal observed in the presence of the inhibitor is indicative of the change in activity experienced by the compound.
  • the change in signal is a change in the signal intensity and/or the signal lifetime. It does not matter whether the change in signal results in a decrease or increase of the signal. Even the loss of any signal is regarded as change in signal.
  • the signal amount or change, respectively, can be then related to the concentration of the inhibitor in the sample, i.e. the calibration curve enables the meter-reading of a matching concentration.
  • the calibration curve is based on the Lambert-Beer equation if using UV/VIS coloring or luminescence.
  • the concentration of the biomarker is subsequently calculated by considering the molar part of MetEEFIA within the product complex if present.
  • the molar ratio of specific substance and MetEEFIA is 1:1 , which is present in antibody/MetEEFIA complexes for instance, so that the molar concentration of the incubation products corresponds to the molar concentration of MetEEFIA.
  • Efficacy of compounds is diagnosed by comparing the concentration of MetEEFIA in the sample with known MetEEFIA concentration levels of either non-treated cells and/or treated cells. It shall be understood that the known concentrations are statistically proven, therefore representing a certain level or range, respectively. Any measured concentration, which differs from the MetEEFIA concentration level of untreated cells, indicates an abnormality of the tested cell sample, whereas a compound cannot be classified as inhibitor at a MetEEFIA concentration that is comparable to the concentration level of untreated cells. It is preferred to measure concentrations, which are higher than the gene product concentration level of untreated cells, for detecting MetAP2 inhibition. Using this method, the inventors demonstrated sensitivity to submicromolar or even nanomolar concentrations. The calibration plot reveals that the method can be applied in a dynamic range that spans over a couple of magnitude.
  • the present screening method is performed such that furthermore in step (c) EEF1A with acetylated N- terminal glycine residue (acGlyEEFIA) is determined, in step (c 1 ) an amount of signal or change in signal is correlated with an acGlyEEFIA concentration in the system and optionally a ratio of MetEEFIA to acGlyEEFIA is determined, and in step (c") the acGlyEEFIA concentration and/or the ratio of MetEEFIA to acGlyEEFIA is compared with another acGlyEEFIA concentration and/or another ratio of MetEEFIA to acGlyEEFIA in a cellular system being not incubated with the compounds and/or in a cellular system being incubated with a standard compound having MetAP2 inhibiting activity.
  • the prior teaching concerning the determination, correlation and detection in view of MetEEFFI A is valid and applicable without restrictions to the detection of acGly
  • the screening method involves another step (d), which comprises the detection of the specific interaction of compounds with a metAP2 gene or a gene product thereof, or a regulator protein, or a component of a signal transduction pathway comprising said gene or a gene product thereof, provided that the aforementioned interaction results in the decrease or even inhibition of MetAP2 activity. It is preferred to detect the specific binding of compounds to the MetAP2 protein target. Preferably, the compounds showing the greatest discrepancy to the control are chosen.
  • the direction and strength of MetEEFIA expression can also been figured out by the differential protein processing analysis of the biomarker of the invention such that either a distinct up-regulation or down-regulation of activity can be recognized, which forms the basis of compound selection.
  • the screening of compounds, which inhibit MetAP2 activity is preferred in the scope of the present invention, the method can also address the screening of MetAP2 activators. More generally, the invention also relates to a method for screening compounds, which alter MetAP2 activity, comprising the steps of: (a) providing a cellular system or a sample thereof being capable of expressing MetAP2, wherein the system is selected from the group of single cells, cell cultures, tissues, organs and mammals,
  • (c 1 ) correlating an amount of signal or change in signal with a MetEEFIA concentration in the system, (c") detecting the MetAP2 activity by comparing the MetEEFIA concentration with another MetEEFIA concentration in a cellular system being not incubated with the compounds and/or in a cellular system being incubated with a standard compound having MetAP2 activating or MetAP2 inhibiting activity, and optionally (d) detecting the specific interaction of compounds, which alter the MetAP2 activity, with a metAP2 gene, or a regulator protein or a gene product thereof, or a component of a signal transduction pathway comprising said gene or a gene product thereof.
  • MetAP2 activation results in the complete conversion of MetEEFIA into acGlyEEFIA (i.e. lack of MetEEFIA), whereas MetAP2 inhibition results in the complete preservation of MetEEFIA (i.e. exclusive appearance of MetEEFIA).
  • the prior teaching of the present specification concerning the method for screening MetAP2 inhibitors is considered as valid and applicable without restrictions to the method for screening MetAP2 modulators if expedient.
  • the prior and ongoing teaching concerning MetAP2 inhibitors is valid and applicable without restrictions to MetAP2 activators if expedient; in doing so it shall be understood for the skilled artisan that certain effects of activating and inhibiting compounds are inherently diametrical.
  • the method of the invention is preferably applied for screening compounds that provide anti-proliferative activity.
  • a proliferative cell culture is applied, such as a small cell lung carcinoma, a non-small cell lung carcinoma, an osteosarcoma, a human breast carcinoma or a contact inhibited mouse fibroblast cell line in particular.
  • MetAP2 inhibitors are highly useful as cytotoxic agents for treating proliferative diseases, preferably cancer including tumors and metastasis. MetAP2 inhibitors are particularly suitable for inhibiting the growth of various lymphomas, sarcomas, carcinomas and myelomas. In addition, MetAP2 inhibitors are suitable for treating angiogenesis-dependent diseases, e.g. various ocular neovascular diseases.
  • the invention also teaches an embodiment of the method for screening therapeutic compounds for an anti-proliferative indication, wherein in step (a) a mammal is provided, in step (b) the compound to be screened are administered to the mammal, and in step (c) a therapeutic effect is detected by determining MetEEFIA in a biological sample withdrawn from the mammal.
  • a mammal is provided
  • step (b) the compound to be screened are administered to the mammal
  • a therapeutic effect is detected by determining MetEEFIA in a biological sample withdrawn from the mammal.
  • the mammal of step (a) is preferably a non-human organism, more preferably a laboratory animal, most preferably species such as mice or rats that may be genetically modified.
  • the mammal suffers from any proliferative clinical picture that is associated with a minor or even absent MetEEFIA level.
  • This expression level of the biomarker MetEEFIA on protein basis is measured in a biopsy sample, such as a tissue sample from tumor tissue or plasma of said mammalian patient, and set as base-line.
  • step (b) it is possible to contact mice or rats, for example, with the compound candidates by injection, infusion, oral or rectal intake. It is preferred to incubate a single compound within a distinct portion of the non-human organisms.
  • Step b) can also be performed in-vitro by exposing ex-vivo a sample, such as a tissue sample from tumor or plasma of the mammalian patient to said anti-MetAP2 drug. A human patient is preferred if performing step (b) in-vitro.
  • step (c) the MetEEFI A expression level is measured again in an identical manner to step a), and differences in the expression levels measured in step a) and c) are calculated.
  • Any difference is inherently based on a change to the initial base-line, thereby confirming an interaction of a defined screening compound with MetAP2.
  • the desired increase in levels of MetEEFI A expression indicates a successful MetAP2 inhibition, which is to be correlated to the therapeutic effect.
  • Such a relationship can be established by monitoring the chronological sequence of the effect against a single or multiple dose of the defined screening compound by using the treated mammal only. The effect is determined either by means of qualitative parameters, e.g. decreasing severity of symptoms, or quantitative parameters, e.g. reduced rate of cell growth rate, diminished tumor size, etc.
  • step (c) An increase in the expression level of the biomarkers MetEEFI A obtained in step (c) compared to step (a) indicates an increased likelihood that said mammal responds therapeutically to the treatment with said screening compound.
  • This increase may approach a threshold under the proviso that the comparison is performed with a healthy control mammal (i.e. lacking any proliferative abnormality), which inherently exhibits a down-regulated MetAP2 activity.
  • the determination of step (c) is favorably performed in comparison with another mammal showing non-proliferative and/or proliferative effects.
  • the comparative mammal is not exposed to compounds to be screened, but treated in an identical manner to measure MetEEFI A levels.
  • Step (c) preferably comprises the further sub-steps of:
  • At least two subjects of a non- human organism suffering of a proliferative disorder are provided as sample, a subset of them the compounds are administered, and the protein processing pattern is correlated to the symptoms of the disorder in subjects to which compounds have been administered and subjects to which no compounds have been administered.
  • step (c) A "therapeutically relevant effect” relieves to some extent one or more symptoms of a disease or returns to normality, either partially or completely, one or more physiological or biochemical parameters associated with or causative of the disease or pathological conditions.
  • therapeutically effective amount denotes an amount which, compared with a corresponding subject who has not received this amount, has the following consequence: improved treatment, healing, prevention or elimination of a disease, syndrome, condition, complaint, disorder or side-effects or also the reduction in the advance of a disease, complaint or disorder.
  • the expression “therapeutically effective amount” also encompasses the amounts which are effective for increasing normal physiological function.
  • the in- vivo dose rate of the chosen compound is advantageously pre-adjusted to the proliferation of the specific cells with regard to their in-vitro data. Therefore, the therapeutic efficacy is remarkably enhanced.
  • a compound identified by the screening method is another object of the invention.
  • the prior teaching of the present specification concerning the screening method is valid and applicable without restrictions to the compound itself if expedient.
  • the identification of compounds that alter the MetAP2 activity, preferably inhibit the MetAP2 activity, which is associated with a given disorder or condition, can lead to the development of pharmaceuticals that can be administered to a patient at therapeutically effective doses to prevent, treat or control such disorder or condition.
  • the invention furthermore relates to a medicament comprising at least one compound according to the invention, and optionally excipients and/or adjuvants.
  • an "adjuvant" denotes every substance that enables, intensifies or modifies a specific response against the active ingredient of the invention if administered simultaneously, contemporarily or sequentially.
  • adjuvants for injection solutions are, for example, aluminum compositions, such as aluminum hydroxide or aluminum phosphate, saponins, such as QS21 , muramyldipeptide or muramyltripeptide, proteins, such as gamma- interferon or TNF, M59, squalen or polyols. Consequently, the invention also relates to a pharmaceutical composition comprising as active ingredient an effective amount of at least one compound as screened according to the invention and/or physiologically acceptable salts thereof together with pharmaceutically tolerable adjuvants.
  • a “medicament”, “drug”, “pharmaceutical composition” or “pharmaceutical formulation” in the meaning of the invention is any agent in the field of medicine, which comprises one or more MetAP2 inhibitors of the invention or preparations thereof and can be used in prophylaxis, therapy, follow-up or aftercare of patients who suffer from diseases, which are associated with MetAP2 signaling, in such a way that a pathogenic modification of their overall condition or of the condition of particular regions of the organism could establish at least temporarily.
  • the active ingredient may be administered alone or in combination with other treatments.
  • a synergistic effect may be achieved by using more than one compound in the pharmaceutical composition, i.e. the MetA2 inhibitor of the invention is combined with at least another agent as active ingredient.
  • the active ingredients can be used either simultaneously or sequentially.
  • compositions can be adapted for administration via any desired suitable method, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods.
  • oral including buccal or sublingual
  • rectal nasal
  • topical including buccal, sublingual or transdermal
  • vaginal or parenteral including subcutaneous, intramuscular, intravenous or intradermal
  • parenteral including subcutaneous, intramuscular, intravenous or intradermal
  • the pharmaceutical composition of the invention is produced in a known way using common solid or liquid carriers, diluents and/or additives and usual adjuvants for pharmaceutical engineering and with an appropriate dosage.
  • the amount of excipient material that is combined with the active ingredient to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Suitable excipients include organic or inorganic substances that are suitable for the different routes of administration, such as enteral (e.g. oral), parenteral or topical application, and which do not react with compounds of the invention or salts thereof.
  • excipients examples include water, vegetable oils, benzyl alcohols, alkylene glycols, polyethylene glycols, glycerol triacetate, gelatin, carbohydrates, such as lactose or starch, magnesium stearate, talc, and petroleum jelly.
  • compositions adapted for oral administration can be administered as separate units, such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or oil-in- water liquid emulsions or water-in-oil liquid emulsions.
  • the active-ingredient component can be combined with an oral, non-toxic and pharmaceutically acceptable inert excipient, such as, for example, ethanol, glycerol, water and the like.
  • Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a pharmaceutical excipient comminuted in a similar manner, such as, for example, an edible carbohydrate, such as, for example, starch or mannitol.
  • a pharmaceutical excipient comminuted in a similar manner, such as, for example, an edible carbohydrate, such as, for example, starch or mannitol.
  • a flavor, preservative, dispersant and dye may likewise be present.
  • suitable binders include starch, gelatin, natural sugars, such as, for example, glucose or beta-lactose, sweeteners made from maize, natural and synthetic rubber, such as, for example, acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • the lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Oral liquids such as, for example, solution, syrups and elixirs, can be prepared in the form of dosage units so that a given quantity comprises a pre-specified amount of the compound.
  • Syrups can be prepared by dissolving the compound in an aqueous solution with a suitable flavor, while elixirs are prepared using a non-toxic alcoholic vehicle.
  • Suspensions can be formulated by dispersion of the compound in a non-toxic vehicle.
  • Solubilisers and emulsifiers such as, for example, ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additives, such as, for example, peppermint oil or natural sweeteners or saccharin, or other artificial sweeteners and the like, can likewise be added.
  • the compounds according to the invention and salts, solvates and physiologically functional derivatives thereof can be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from various phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • the active ingredient according to the invention can also be fused or complexed with another molecule that promotes the directed transport to the destination, the incorporation and/or distribution within the target cells.
  • the compounds according to the invention and the salts, solvates and physiologically functional derivatives thereof can also be delivered using monoclonal antibodies as individual carriers to which the compound molecules are coupled.
  • the compounds can also be coupled to soluble polymers as targeted medicament carriers.
  • Such polymers may encompass polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamido- phenol, polyhydroxyethylaspartamidophenol or polyethylene oxide polylysine, substituted by palmitoyl radicals.
  • the compounds may furthermore be coupled to a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • compositions adapted for transdermal administration can be administered as independent plasters for extended, close contact with the epidermis of the recipient.
  • the active ingredient can be delivered from the piaster by iontophoresis, as described in general terms in Pharmaceutical Research, 3(6), 318 (1986).
  • compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the formulations are preferably applied as topical ointment or cream.
  • the active ingredient can be employed either with a paraffinic or a water-miscible cream base.
  • the active ingredient can be formulated to give a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions adapted for topical application to the eye include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent.
  • Pharmaceutical formulations adapted for topical application in the mouth encompass lozenges, pastilles and mouthwashes.
  • compositions adapted for rectal administration can be administered in the form of suppositories or enemas.
  • compositions adapted for nasal administration in which the carrier substance is a solid comprise a coarse powder having a particle size, for example, in the range 20-500 microns, which is administered in the manner in which snuff is taken, i.e. by rapid inhalation via the nasal passages from a container containing the powder held close to the nose.
  • suitable formulations for administration as nasal spray or nose drops with a liquid as carrier substance encompass active-ingredient solutions in water or oil.
  • Pharmaceuticai formulations adapted for administration by inhalation encompass finely particulate dusts or mists, which can be generated by various types of pressurized dispensers with aerosols, nebulisers or insufflators.
  • compositions adapted for vaginal administration can be administered as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions comprising antioxidants, buffers, bacteriostatics and solutes, by means of which the formulation is rendered isotonic with the blood of the recipient to be treated; and aqueous and non-aqueous sterile suspensions, which may comprise suspension media and thickeners.
  • the formulations can be administered in single-dose or multi-dose containers, for example sealed ampoules and vials, and stored in freeze-dried (lyophilized) state, so that only the addition of the sterile carrier liquid, for example water for injection purposes, immediately before use is necessary.
  • Injection solutions and suspensions prepared in accordance with the recipe can be prepared from sterile powders, granules and tablets.
  • formulations may also comprise other agents usual in the art with respect to the particular type of formulation; thus, for example, formulations which are suitable for oral administration may comprise flavors.
  • the pharmaceutical composition is orally or parenterally administered, more preferably orally.
  • the active ingredient is provided in a water-soluble form, such as a pharmaceutically acceptable salt, which is meant to include both acid and base addition salts.
  • the compounds of the invention and salts thereof may be lyophilized and the resulting lyophilizates used, for example, to produce preparations for injection.
  • the preparations indicated may be sterilized and/or may comprise auxiliaries, such as carrier proteins (e.g.
  • compositions can be administered in the form of dosage units which comprise a predetermined amount of active ingredient per dosage unit.
  • concentration of the prophylactically or therapeutically active ingredient in the formulation may vary from about 0.1 to 100 wt %.
  • the compound of formula (I) or the pharmaceutically acceptable salts thereof are administered in doses of approximately 0.5 to 1000 mg, more preferably between 1 and 700 mg, most preferably 5 and 100 mg per dose unit. Generally, such a dose range is appropriate for total daily incorporation. In other terms, the daily dose is preferably between approximately 0.02 and 100 mg/kg of body weight.
  • the specific dose for each patient depends, however, on a wide variety of factors (e.g. depending on the condition treated, the method of administration and the age, weight and condition of the patient).
  • Preferred dosage unit formulations are those which comprise a daily dose or part-dose, as indicated above, or a corresponding fraction thereof of an active ingredient.
  • pharmaceutical formulations of this type can be prepared using a process which is generally known in the pharmaceutical art.
  • the invention also relates to a method for monitoring physiological and/or pathological conditions, which are caused, mediated and/or propagated by MetAP2 activity, wherein an effective amount of at least one compound or a physiologically acceptable salt thereof is administered to a mammal in need of such treatment and MetEEFIA is determined in a biological sample withdrawn from the mammal.
  • MetEEFIA is determined in a biological sample withdrawn from the mammal.
  • An increase in the expression level of said biomarker over the period of monitoring indicates a decreased MetAP2 activity, which is associated with an increased likelihood that said mammal responds to the treatment with said compound.
  • the compound is preferably obtained by the screening method of the invention as set forth above.
  • EEF1A provides a powerful tool for assessing the progression of a state, condition or treatment.
  • the present invention can be used as a clinical marker to monitor efficacy of a MetAP2 inhibitor compound on each patient individually.
  • EEF1A can be identified in a patient prior to an event, such as menopause, surgery, the onset of a therapeutic regime, or the completion of a therapeutic regime, to provide a base line result. This base-line can then be compared with the result obtained using identical methods either during or after such event.
  • This information can be used for both diagnostic and prognostic purposes.
  • the information about the clinical marker can be additionally used to optimize the dosage and the regimen of an active compound by monitoring the induction and accumulation of MetEEFIA in the subject's biological sample.
  • the method of the present invention can be used to find a therapeutically effective compound and/or a therapeutically effective amount or regimen for the selected compound, thereby individually selecting and optimizing a therapy for a patient.
  • the inventive method of monitoring can be employed in human and veterinary medicine.
  • the mammal is preferably a laboratory animal and/or a non-human organism.
  • the compounds can be administered before or following an onset of disease once or several times acting as therapy.
  • the terms "effective amount” or “effective dose” or “dose” are interchangeably used herein and denote an amount of the pharmaceutical compound having a prophylactically or therapeutically relevant effect on a disease or pathological conditions, i.e. which causes in a tissue, system, animal or human a biological or medical response which is sought or desired, for example, by a researcher or physician.
  • the aforementioned medical products of the inventive use are particularly used for the therapeutic treatment.
  • Monitoring is considered as a kind of treatment, wherein the compounds are preferably administered in distinct intervals, e.g. in order to booster the response and eradicate the pathogens and/or symptoms of the MetAP2-related disease completely. Either the identical compound or different compounds can be applied.
  • the medicament can also be used to reduce the likelihood of developing a disease or even prevent the initiation of diseases associated with MetAP2 activity in advance or to treat the arising and continuing symptoms.
  • prophylactic treatment is advisable if the subject possesses any preconditions for the aforementioned physiological or pathological conditions, such as a familial disposition, a genetic defect, or a previously passed disease.
  • the diseases as concerned by the invention are cancerous diseases, which are selected from the group of cancerous diseases of the ear-nose-throat region, the lungs, mediastinum, gastrointestinal tract, urogenital system, gynecological system, breast, endocrine system, skin and bone, furthermore soft-tissue sarcomas, mesotheliomas, melanomas, neoplasms of the central nervous system, cancerous diseases during infancy, lymphomas, leukemias, paraneoplastic syndromes, metastases with unknown primary tumor (CUP syndrome), peritoneal carcinomatoses, immunosuppression-related malignancies and/or tumor metastases.
  • cancerous diseases which are selected from the group of cancerous diseases of the ear-nose-throat region, the lungs, mediastinum, gastrointestinal tract, urogenital system, gynecological system, breast, endocrine system, skin and bone, furthermore soft-tissue sarcomas, mesot
  • Walker carcinoma basal cell carcinoma, squamobasal carcinoma, Brown-Pearce carcinoma, ductal carcinoma, Ehrlich tumor, in-situ carcinoma, cancer-2 carcinoma, Merkel cell carcinoma, mucous cancer, non-parvicellular bronchial carcinoma, oat-cell carcinoma, papillary carcinoma, scirrhus carcinoma, bronchio-alveolar carcinoma, bronchial carcinoma, squamous cell carcinoma and transitional cell carcinoma), histiocytic functional disorder, leukemia (e.g.
  • B cell leukemia in connection with B cell leukemia, mixed-cell leukemia, null cell leukemia, T cell leukemia, chronic T cell leukemia, HTLV-I l-associated leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, mast cell leukemia, and myeloid leukemia), malignant histiocytosis, Hodgkin disease, non-Hodgkin lymphoma, solitary plasma cell tumor; reticuloendotheliosis, chondroblastoma, " chondroma, chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors, histiocytoma, lipoma, liposarcoma, leukosarcoma, mesothelioma, myxoma, myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma, adenofibrom
  • the tumor is preferably selected from the group of tumors of the squamous epithelium, the bladder, the stomach, the kidneys, the head, the neck, the oesophagus, the cervix, the thyroid, the intestine, the liver, the brain, the prostate, the urogenital tract, the lymphatic system, the stomach, the larynx and/or the lung.
  • the tumor is furthermore preferably selected from the group of lung adenocarcinoma, small-cell lung carcinomas, pancreatic cancer, glioblastomas, colon carcinoma and breast carcinoma.
  • preference is given to the treatment and/or monitoring of a tumor of the blood and immune system, more preferably for the treatment and/or monitoring of a tumor selected from the group of acute myeloid leukemia, chronic myeloid leukemia, acute lymphatic leukemia and/or chronic lymphatic leukemia.
  • Such tumors can also be designated as cancers in the meaning of the invention.
  • the said compounds according to the invention can be used in their final non-salt form.
  • the present invention also encompasses the use of these compounds in the form of their pharmaceutically acceptable salts, which can be derived from various organic and inorganic acids and bases by procedures known in the art.
  • pharmaceutically acceptable salt and “physiologically acceptable salt”, which are used interchangeable herein, in the present connection are taken to mean an active ingredient which comprises a compound according to the invention in the form of one of its salts, in particular if this salt form imparts improved pharmacokinetic properties on the active ingredient compared with the free form of the active ingredient or any other salt form of the active ingredient used earlier.
  • the invention also relates to a method for determining MetAP2 activity comprising the steps of: (a) providing a cellular system or a sample thereof being capable of expressing MetAP2, wherein the system is selected from the group of single cells, cell cultures, tissues, organs and mammals,
  • any partial activity between complete inhibition or maximal activity can be set or even the aforementioned thresholds can be achieved.
  • the inverse proportionality can follow either a linear or a non-linear function.
  • the prior teaching concerning the methods for screening compounds as well as monitoring physiological and/or pathological conditions is valid and applicable without restrictions to the method for determining MetAP2 activity.
  • the invention relates to an in-vitro method for predicting the likelihood that a patient suffering from a tumor, who is a candidate for treatment with an anti-MetAP2 drug, will respond to the treatment with said drug, comprising the determination of the expression level of a prognostic gene expression product, which is MetEEFIA, in a tissue sample obtained from said patient, wherein a higher expression of the gene product indicates that the patient is likely to respond to said treatment compared to a reference value.
  • the treatment with said anti-MetAP2 drug can be a first-line mono-therapy and the selected gene expression product is the expressed protein MetEEFIA.
  • the treatment with said anti-MetAP2 drug is a combination therapy with a chemotherapeutic agent after the patient has developed a chemo-refractory tumor, and the selected gene expression product is the expressed protein MetEEFIA.
  • the reference value is defined by one ore more of a specific functional or clinical property, and/or a specific, genetic or protein expression profile obtained from a reference patient or reference patient group. Said reference patient or patient group that does not express or express less gene product compared to the candidate patient.
  • the reference value is an expression threshold value which is individually constituted or defined by specific clinical response parameters to be determined or by specific pre-treatment or treatment conditions.
  • Suitable clinical response parameters are the progression free survival time (PFS), overall survival time (OS), partial response (PR), stable response (SR), progressive disease (PD) or combinations thereof.
  • Tissue samples are taken from the patient before treatment with said anti-MetAP2 drug and additionally on treatment with said anti-MetAP2 drug.
  • the expression levels of the gene expression product obtained on treatment are compared with the values obtained before starting treatment of said patient.
  • the patient sample derives from tumor tissue or plasma, for example.
  • Another in-vitro method for predicting the likelihood that a patient suffering from a proliferative-driven disorder or condition, particularly cancer, will respond therapeutically to the treatment with an anti-MetAP2 drug comprises the steps of (a) measuring in a biopsy tissue sample from tumor tissue or plasma of said patient the expression level of MetEEFIA biomarker on protein basis, (b) exposing ex-vivo a tissue sample from tumor or plasma of said patient to said anti-MetAP2 drug, and (c) measuring in said exposed tissue sample of step (b) the expression level of said biomarker specified in step (a) along with calculating the differences in expression levels measured in steps (b) and (c), wherein an increase in the expression level of said biomarker obtained in this step (c) compared to step (a) indicates an increased likelihood that said patient responds therapeutically to the treatment with said anti-MetAP2 drug.
  • Object of the invention is also the use of EEF1A1 comprising the amino acid sequence of SEQ ID NO: 1 and/or EEF1A2 comprising the amino acid sequence of SEQ ID NO: 2 (cf. Figure 11), or variants, mutants, parts of the amino acid sequence or at least 85 % homologous sequences having the same function, or a nucleic acid encoding EEF1A1 and/or a nucleic acid sequence encoding EEF1A2, as biomarker for a reduction of MetAP2 activity.
  • Another object of the invention relates to the use of EEF1 A as biomarker for cell proliferation under the proviso that oral tongue squamous cell carcinoma, head squamous cell carcinoma, neck squamous cell carcinoma, pancreas cancer and breast cancer are disclaimed. It is still another object of the invention to use EEF1A as biomarker for a reduction of likelihood of developing a tumor and/or progressive tumor growth under the proviso that breast cancer is disclaimed. A preferred use concerns the application of EEF1 A as biomarker for angiogenic and/or tumor inhibition.
  • the biomarker can be used for monitoring, determining and/or predicting the reduction of MetAP2 activity, the status of cell proliferation and/or the reduction of likelihood of developing a tumor and/or a progressive tumor growth.
  • the different uses can be subsumed under the general term "assessing”. It goes without saying that data are monitored over a specific period, while data are determined at a particular time. Both the period and the time can be easily designed by the skilled artisan depending on the experimental trials conducted.
  • EEF1A can be used as biomarker for predicting in-vitro the pharmaceutical efficacy and/or clinical response of a mammal suffering from cancer to a MetAP2-inhibiting drug, which is intended to be administered and/or is administered in cancer treatment.
  • the underlying treatment is particularly a first-line treatment, and the inhibitory drug with which the mammalian patient is to be treated, is administered in mono-therapy.
  • said drug is combined with a chemotherapeutic agent, and said patient has developed chemo-refractory cancer.
  • MetEEF1A1 and/or MetEEF1A2 are preferred use concerns that of MetEEF1A1 and/or MetEEF1A2 as biomarker.
  • the prior teaching of the present specification concerning the screening method is valid and applicable without restrictions to any of said uses if expedient.
  • the invention may be practiced as a kit comprising substances specifically interacting with EEF 1 A, preferably MetEEFIA, particularly in order to perform the inventive method for detecting and/or characterizing MetAP2 activity.
  • the kit of the invention may include an article that comprises written instructions or directs the user to written instructions for how to practice the method of the invention.
  • the kit further comprises a reporter moiety or a reporter apparatus.
  • the kit may comprise an extracting reagent for isolating the biomarker.
  • a method for screening MetAP2 inhibitors or modulators by applying the unique biomarker EEF1 A is provided for the first time.
  • the present invention teaches the induction and accumulation of MetEEFIA in cells, whose enzymatic MetAP2 activity is interrupted.
  • EEF1A as a biomarker for MetAP2 inhibitors has several advantages. It is an abundant cellular protein so it can be easily detected in tumor samples. The N-terminal status of EEF1A reflects the MetAP2 enzyme activity over a period, thus truly representing the consequence of MetAP2 inhibition and better correlating with efficacy of the testing compound.
  • MetEEFIA readout is on MetAP2-specific substrate, but not MetAP2 itself, so it can be used for all types of MetAP2 inhibitors.
  • the substrate is processed by MetAP2, but not by MetAPI , which makes it possible to prove that an anti-angiogenic and anti-proliferative activity is specifically due to inhibition of the proteolytic activity of MetAP2.
  • lnhibition of methionine processing by MetAP2 blocks tumor cell growth in-vitro. MetAP2 inhibitors even induce MetEEFI A in a dose-dependent fashion similar to that observed for their antiproliferative activity.
  • the robust marker has the potential to monitor MetAP2 inhibition not only in cells but also in animals and human subjects treated with MetAP2 inhibitors.
  • EEF1A N-terminal status is an outstanding biomarker for cellular MetAP2 inhibition in-vitro and in-vivo. EEF1A processing is of benefit to monitor MetAP2 inhibition in preclinical and clinical testing.
  • the analysis of the differential processed EEF1A isoform is very suitable for large-scale screening tests.
  • the novel marker allows the identification of novel MetAP2 inhibitors.
  • Compounds can be identified and evaluated with a specific cellular mechanism of action and additionally, their potential to exert anti-angiogenic and/or anti-proliferative effects can be favorably proved.
  • the characterization of EEF1A, particularly MetEEFIA, critically involved in substrate recognition by MetAP2 results in the provision of pharmaceutical compositions for the diagnosis, prophylactic or therapeutic treatment and/or monitoring of conditions, which are caused, mediated and/or propagated by MetAP2 activity.
  • Their use is a promising, novel approach for a broad spectrum of therapies causing a direct and immediate reduction of symptoms that are clearly connected with MetAP2-dependent diseases.
  • the compounds are of special benefit as anti-cancer agents in mammals.
  • Human fibrosarcoma cells (HT1080, ATCC CCL-121) were cultured at 37°C and 10 % CO 2 Jn D-MEM (Invitrogen, #41965) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Pan Biotech, #3302).
  • IEF gels (CleanGel IEF ultra ETC1001-52 from ETC, Germany) were re-hydrated in 7 M urea, 2 M thiourea, 1 % DTE, 4 % CHAPS, 3 %, Servalyte 7-9 for 120 min, and assembled on a Multiphor Il electrophoresis unit (GE Healthcare). Samples (5 ⁇ l) were applied close to the anode and run with anode buffer (6 M glycine) and cathode fluid 10 (Serva, Germany).
  • Coomassie Blue stained acrylamide gel bands were excised after IEF separation, washed twice with 50 % acetonitrile and rinsed with pure acetonitrile.
  • In gel digest was performed by adding 0.1 ⁇ g trypsin dissolved in 10 ⁇ l ammonium bicarbonate pH 7.4 to the gel band. Incubation was achieved at 37°C overnight. Aliquots of the digest mix were applied onto a target plate of a MALDI-TOF MS (Ultraflex, Bruker, Germany) and ⁇ -cyano-4-hydroxycinnamic acid was added as a matrix. Mass analysis was performed and peptide fragments were detected applying standard protocols.
  • the achieved peptide mass lists were searched against the SwissProt database using the Mascot search algorithm (Matrix Science, UK) to identify the protein. Furthermore, distinct peptides from a mass map were used for subsequent MS-fragmentation studies in order to determine the primary sequence of those individual peptides and for unambiguous protein annotation.
  • Table 1 contains explanatory information for Figure 10.
  • Compounds were incubated with bEND3 cells for 48 h at the indicated concentrations, lysed and electrophoretic mobility shift of EEF1A1 was assessed in an IEF gel.
  • the compounds used were characterized as inhibitors of MetAP2 and inhibitors of HUVEC cell proliferation with the indicated potencies (IC50).
  • Inhibition of MetAP2 was measured via cleavage of the tripeptide Met-Ala-Ser and subsequent detection of cleaved methionine using an enzyme-coupling reaction composed of L-amino oxidase and peroxidase (Wang et al. (2003) Biochemistry_42(17): 5035-5042).
  • Figure 1 shows the treatment-related pattern of cellular extracts from different endothelial and cancer cells after IEF separation with a new band observed after applying MetAP2-inhibitors (lane 1 , 5, 9: IEF marker proteins; lane 2, 6, 12: 100 nM TNP470; lane 3, 7:
  • Figure 2 shows a zoomed view for lanes 2-4 from Figure 1.
  • Figure 3 shows the results of Edman sequencing that confirms Met 1 at the N-terminus of EEF1A1 after MetAP2 inhibition. Sequence detected for TNP470-treated HT1080 + bEND3 cells is M 1 GKEKTHINI.
  • Figure 5 shows the determination of N-terminus comprising the steps of: localization of N-terminal peptide, sequence determination by MSMS and characterization of N- terminal processing.
  • the mass map of Band 1 corresponds to the sequence acGKEKTHINIWIGHVDSGK (M+H + : 2.073,2); the mass map of Band 2 corresponds to the sequence MGKEKTHINIWIGHVDSGK (M+H + : 2.162,2).
  • Figure 6 shows the MSMS analysis of parent mass 2.073,2 D from the tryptic digest of the Band 1 , which reveals the sequence acGKEKTHINIWIGHVDSGK.
  • Figure 7 shows the MSMS analysis of parent mass 2.162,2 D from the tryptic digest of the Band 2, which reveals the sequence MGKEKTHINIWIGHVDSGK.
  • Figure 8 shows the evaluation of the EEF1A1 identification for two cell extracts (lane 1, 3: DMSO; lane 2, 4: TNP470).
  • Figure 10 shows the EEF1A1 mobility shift with negative controls unequivocally distinguishable from active MetAP2 inhibitors (TNP-470, A-832234; lane 1-4: cf. Table 1).
  • Figure 11 shows the sequence alignment of EEF1A1 (P68104; SEQ ID NO: 1) and EEF1A2 (Q05639; SEQ ID NO: 2), which has 92.7 % sequence identity. Both accession numbers refer to the sequence database Swiss-Prot.
  • the two isoforms EEF1 A1 and EEF1A2 comprise a sequence identity of 92.7 % ( Figure 11). Both isoforms are identical within the first 82 amino acids and, therefore, are substrates of MetAP2.

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Abstract

La présente invention concerne un procédé permettant le criblage de composés inhibiteurs de l’activité de MetAP2, par la fourniture d’un système cellulaire ou d’un échantillon de celui-ci capable d’exprimer MetAP2 et/ou eEF1A,, l’incubation d’au moins une partie du système avec les composés à cribler, et la détection d’inhibition de MetAP2 par la détermination d’EEF1A avec un résidu de méthionine à l’extrémité N (MetEEFIA). L’invention concerne également un procédé permettant le contrôle de conditions physiologiques et/ou pathologiques, qui sont provoquées, médiées et/ou propagées par l’activité de MetAP2, par l’administration d’une quantité efficace d’au moins un composé unique à un mammifère qui a besoin d’un tel traitement et la détermination de MetEEFIA dans un échantillon biologique prélevé du mammifère. L’invention concerne également l’utilisation d’EEF1A comme biomarqueur.
PCT/EP2009/007102 2008-11-06 2009-10-05 Utilisation d’eef1a comme biomarqueur et procédé de criblage d’inhibiteurs de metap2 WO2010051882A1 (fr)

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ES09778819T ES2402564T3 (es) 2008-11-06 2009-10-05 Uso de EEF1A como biomarcador para el cribado de inhibidores de METAP2
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AU2009313167A AU2009313167C1 (en) 2008-11-06 2009-10-05 Use of EEF1A as biomarker for screening of MetAP2 inhibitors
US13/127,834 US9151743B2 (en) 2008-11-06 2009-10-05 Use of EEF1A as biomarker and a method of screening MetAP2 inhibitors
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CN107921149A (zh) * 2015-07-31 2018-04-17 高丽大学校产学协力团 非酒精性脂肪肝调控因子14‑3‑3蛋白
CN107921149B (zh) * 2015-07-31 2021-10-15 高丽大学校产学协力团 非酒精性脂肪肝调控因子14-3-3蛋白
WO2018148652A1 (fr) * 2017-02-10 2018-08-16 Zafgen, Inc. Procédés d'identification de modulateurs de metap-2

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EP2344665A1 (fr) 2011-07-20
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US20110275088A1 (en) 2011-11-10
JP5347028B2 (ja) 2013-11-20
CA2742737C (fr) 2018-05-29
ES2402564T3 (es) 2013-05-06
AU2009313167C1 (en) 2014-06-12
IL212664A0 (en) 2011-07-31
CA2742737A1 (fr) 2010-05-14
AU2009313167A1 (en) 2011-06-23
JP2012507268A (ja) 2012-03-29

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